The long-term objective is to determine the cellular mechanism of transport of inorganic phosphate (Pi) by the renal proximal tubule. The specific aims are: 1) to define the role of gluconeogenesis (GNG) in regulation of Pi transport acress luminal brush border membrane (BBM); 2) to determine the mechanism by which a change in the level of NAD+ in cytosol, mediated by a change in GNG, leads to modification of BBM transport of Pi. Filtered Pi is reabsorbed principally in the proximal tubule which is the main site of GNG in the kidney. The GNG pathway is of particular interest for a possible role in regulation of Pi transport because the rate of GNG is increased by phosphaturic stimuli and is decreased by antiphosphaturic stimuli. The mechanism of regulation may be via the effect of GNG on the NAD+/NADH ratio in cytosol since NAD+ inhibits Pi transport by renal BBM and the amount of NAD+ in cytosol will be changed by a switch in the NAD+/NADH ratio. In cytosol most of the NADH is protein-bound but most of the NAD+ exists free and is available to interact with the BBM. Using tissue slices or renal tubule suspensions the experiments will determine whether changes in GNG lead to changes in Pi transport, and whether the action of phosphaturic and antiphosphaturic stimuli on Pi transport is dependent on the effect of these stimuli on GNG. Other experiments will investigate the mechanism by which interaction of NAD+ with the BBM leads to changes in BBM transport of Pi. The rate of GNG will be determined by measuring glucose production by slices of renal cortex or suspensions of proximal tubules. Renal BBM transport of Pi will be assessed by measuring 32Pi uptake by isolated renal BBM vesicles. Covalent modification of BBM proteins by NAD+ will be determined from acid-stable binding of radiolabelled NAD+. These studies will provide a fundamental basis of the study of renal disease involving altered renal handling of Pi.